Family D DNA polymerase interacts with GINS to promote CMG-helicase in the archaeal replisome

Abstract Genomic DNA replication requires replisome assembly. We show here the molecular mechanism by which CMG (GAN–MCM–GINS)-like helicase cooperates with the family D DNA polymerase (PolD) in Thermococcus kodakarensis. The archaeal GINS contains two Gins51 subunits, the C-terminal domain of which (Gins51C) interacts with GAN. We discovered that Gins51C also interacts with the N-terminal domain of PolD’s DP1 subunit (DP1N) to connect two PolDs in GINS. The two replicases in the replisome should be responsible for leading- and lagging-strand synthesis, respectively. Crystal structure analysis of the DP1N–Gins51C–GAN ternary complex was provided to understand the structural basis of the connection between the helicase and DNA polymerase. Site-directed mutagenesis analysis supported the interaction mode obtained from the crystal structure. Furthermore, the assembly of helicase and replicase identified in this study is also conserved in Eukarya. PolD enhances the parental strand unwinding via stimulation of ATPase activity of the CMG-complex. This is the first evidence of the functional connection between replicase and helicase in Archaea. These results suggest that the direct interaction of PolD with CMG-helicase is critical for synchronizing strand unwinding and nascent strand synthesis and possibly provide a functional machinery for the effective progression of the replication fork.

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Critical residues for ligand binding in blade 2 of the propeller domain of the integrin αIIb subunit

Thrombosis and Haemostasis ◽

10.1160/th03-06-0392 ◽

2004 ◽

Vol 91 (01) ◽

pp. 111-118 ◽

Cited By ~ 4

Author(s):

Tatsushiro Tamura ◽

Jun Yamanouchi ◽

Shigeru Fujita ◽

Takaaki Hato

Keyword(s):

Crystal Structure ◽

Ligand Binding ◽

Binding Site ◽

Thrombus Formation ◽

Direct Interaction ◽

Binding Pocket ◽

Crystal Structure Analysis ◽

Site Directed Mutagenesis ◽

Structural Basis ◽

Critical Residues

SummaryLigand binding to integrin αIIbβ3 is a key event of thrombus formation. The propeller domain of the αIIb subunit has been implicated in ligand binding. Recently, the ligand binding site of the αV propeller was determined by crystal structure analysis. However, the structural basis of ligand recognition by the αIIb propeller remains to be determined. In this study, we conducted site-directed mutagenesis of all residues located in the loops extending above blades 2 and 4 of the αIIb propeller, which are spatially close to, but distinct from, the loops that contain the binding site for an RGD ligand in the crystal structure of the αV propeller. Replacement by alanine of Q111, H112 or N114 in the loop within the blade 2 (the W2:2-3 loop in the propeller model) abolished binding of a ligand-mimetic antibody and fibrinogen to αIIbβ3 induced by different types of integrin activation including activation of αIIbβ3 by β3 cytoplasmic mutation. CHO cells stably expressing recombinant αIIbβ3 bearing Q111A, H112A or N114A mutation did not exhibit αIIbβ3mediated adhesion to fibrinogen. According to the crystal structure of αVβ3, the αV residue corresponding to αIIbN114 is exposed on the integrin surface and close to the RGD binding site. These results suggest that the Q111, H112 and N114 residues in the loop within blade 2 of the αIIb propeller are critical for ligand binding, possibly because of direct interaction with ligands or modulation of the RGD binding pocket.

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Structural and biochemical characterization of bifunctional XynA

10.1101/2020.10.20.348094 ◽

2020 ◽

Author(s):

Wei Xie ◽

Qi Yu ◽

Yun Liu ◽

Ruoting Cao ◽

Ruiqing Zhang ◽

...

Keyword(s):

Crystal Structure ◽

Enzyme Activity ◽

Catalytic Mechanism ◽

Biochemical Characterization ◽

Cellulase Activity ◽

Cost Savings ◽

Site Directed Mutagenesis ◽

Enzyme Activity Assay ◽

Great Cost ◽

Terminal Domain

AbstractXylan and cellulose are the two major constituents in numerous types of lignocellulosic biomass, representing a promising resource for biofuels and other biobased industries. The efficient degradation of lignocellulose requires the synergistic actions of cellulase and xylanase. Thus, bifunctional enzyme incorporated xylanase/cellulase activity has attracted considerable attention since it has great cost savings potential. Recently, a novel GH10 family enzyme XynA identified from Bacillus sp. is found to degrade both cellulose and xylan. To understand its molecular catalytic mechanism, here we first solve the crystal structure of XynA at 2.3 Å. XynA is characterized with a classic (α/β)8 TIM-barrel fold (GH10 domain) flanked by the flexible N-terminal domain and C-terminal domain. Circular dichroism, protein thermal shift and enzyme activity assays reveal that conserved residues Glu182 and Glu280 are both important for catalytic activities of XynA, which is verified by the crystal structure of XynA with E182A/E280A double mutant. Molecular docking studies of XynA with xylohexaose and cellohexaose as well as site-directed mutagenesis and enzyme activity assay demonstrat that Gln250 and His252 are indispensible to cellulase and bifunctional activity, separately. These results elucidate the structural and biochemical features of XynA, providing clues for further modification of XynA for industrial application.

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Crystal structure of the complex of the interaction domains of Escherichia coli DnaB helicase and DnaC helicase loader: structural basis implying a distortion-accumulation mechanism for the DnaB ring opening caused by DnaC binding

The Journal of Biochemistry ◽

10.1093/jb/mvz087 ◽

2019 ◽

Vol 167 (1) ◽

pp. 1-14

Author(s):

Koji Nagata ◽

Akitoshi Okada ◽

Jun Ohtsuka ◽

Takatoshi Ohkuri ◽

Yusuke Akama ◽

...

Keyword(s):

Crystal Structure ◽

Escherichia Coli ◽

Molecular Model ◽

Structural Basis ◽

Terminal Domain ◽

Helicase Dnab ◽

Accumulation Mechanism ◽

Interaction Domains ◽

Α Helix ◽

Available Information

Abstract Loading the bacterial replicative helicase DnaB onto DNA requires a specific loader protein, DnaC/DnaI, which creates the loading-competent state by opening the DnaB hexameric ring. To understand the molecular mechanism by which DnaC/DnaI opens the DnaB ring, we solved 3.1-Å co-crystal structure of the interaction domains of Escherichia coli DnaB–DnaC. The structure reveals that one N-terminal domain (NTD) of DnaC interacts with both the linker helix of a DnaB molecule and the C-terminal domain (CTD) of the adjacent DnaB molecule by forming a three α-helix bundle, which fixes the relative orientation of the two adjacent DnaB CTDs. The importance of the intermolecular interface in the crystal structure was supported by the mutational data of DnaB and DnaC. Based on the crystal structure and other available information on DnaB–DnaC structures, we constructed a molecular model of the hexameric DnaB CTDs bound by six DnaC NTDs. This model suggested that the binding of a DnaC would cause a distortion in the hexameric ring of DnaB. This distortion of the DnaB ring might accumulate by the binding of up to six DnaC molecules, resulting in the DnaB ring to open.

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Crystal Structure of Classical Swine Fever Virus NS5B Reveals a Novel N-Terminal Domain

Journal of Virology ◽

10.1128/jvi.00324-18 ◽

2018 ◽

Vol 92 (14) ◽

Cited By ~ 5

Author(s):

Weiwei Li ◽

Baixing Wu ◽

Wibowo Adian Soca ◽

Lei An

Keyword(s):

Crystal Structure ◽

Rna Polymerase ◽

Classical Swine Fever Virus ◽

Classical Swine Fever ◽

Fever Virus ◽

Structural Basis ◽

Economic Losses ◽

Rna Dependent Rna Polymerase ◽

Content Type ◽

Terminal Domain

ABSTRACTClassical swine fever virus (CSFV) is the cause of classical swine fever (CSF). Nonstructural protein 5B (NS5B) is an RNA-dependent RNA polymerase (RdRp) that is a key enzyme initiating viral RNA replication by ade novomechanism. It is also an attractive target for the development of anti-CSFV drugs. To gain a better understanding of the mechanism of CSFV RNA synthesis, here, we solved the first crystal structure of CSFV NS5B. Our studies show that the CSFV NS5B RdRp contains the characteristic finger, palm, and thumb domains, as well as a unique N-terminal domain (NTD) that has never been observed. Mutagenesis studies on NS5B validated the importance of the NTD in the catalytic activity of this novel RNA-dependent RNA polymerase. Moreover, our results shed light on CSFV infection.IMPORTANCEPigs are important domesticated animals. However, a highly contagious viral disease named classical swine fever (CSF) causes devastating economic losses. Classical swine fever virus (CSFV), the primary cause of CSF, is a positive-sense single-stranded RNA virus belonging to the genusPestivirus, familyFlaviviridae. Genome replication of CSFV depends on an RNA-dependent RNA polymerase (RdRp) known as NS5B. However, the structure of CSFV NS5B has never been reported, and the mechanism of CSFV replication is poorly understood. Here, we solve the first crystal structure of CSFV NS5B and analyze the functions of the characteristic finger, palm, and thumb domains. Additionally, our structure revealed the presence of a novel N-terminal domain (NTD). Biochemical studies demonstrated that the NTD of CSFV NS5B is very important for RdRp activity. Collectively, our studies provide a structural basis for future rational design of anti-CSFV drugs, which is critically important, as no effective anti-CSFV drugs have been developed.

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Structural Basis of Ubiquitin Recognition by the Ubiquitin-associated (UBA) Domain of the Ubiquitin Ligase EDD

Journal of Biological Chemistry ◽

10.1074/jbc.m705655200 ◽

2007 ◽

Vol 282 (49) ◽

pp. 35787-35795 ◽

Cited By ~ 39

Author(s):

Guennadi Kozlov ◽

Long Nguyen ◽

Tong Lin ◽

Gregory De Crescenzo ◽

Morag Park ◽

...

Keyword(s):

Ubiquitin Ligase ◽

Site Directed Mutagenesis ◽

Structural Basis ◽

Titration Calorimetry ◽

Polyubiquitin Chains ◽

C Terminus ◽

Damage Repair ◽

The Family ◽

Uba Domain ◽

Ordered Water

EDD (or HYD) is an E3 ubiquitin ligase in the family of HECT (homologous to E6-AP C terminus) ligases. EDD contains an N-terminal ubiquitin-associated (UBA) domain, which is present in a variety of proteins involved in ubiquitin-mediated processes. Here, we use isothermal titration calorimetry (ITC), NMR titrations, and pull-down assays to show that the EDD UBA domain binds ubiquitin. The 1.85Å crystal structure of the complex with ubiquitin reveals the structural basis of ubiquitin recognition by UBA helices α1 and α3. The structure shows a larger number of intermolecular hydrogen bonds than observed in previous UBA/ubiquitin complexes. Two of these involve ordered water molecules. The functional importance of residues at the UBA/ubiquitin interface was confirmed using site-directed mutagenesis. Surface plasmon resonance (SPR) measurements show that the EDD UBA domain does not have a strong preference for polyubiquitin chains over monoubiquitin. This suggests that EDD binds to monoubiquitinated proteins, which is consistent with its involvement in DNA damage repair pathways.

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Crystal structure analysis of Bacillus subtilis ferredoxin-NADP+ oxidoreductase and the structural basis for its substrate selectivity

Protein Science ◽

10.1002/pro.508 ◽

2010 ◽

Vol 19 (12) ◽

pp. 2279-2290 ◽

Cited By ~ 18

Author(s):

Hirofumi Komori ◽

Daisuke Seo ◽

Takeshi Sakurai ◽

Yoshiki Higuchi

Keyword(s):

Crystal Structure ◽

Bacillus Subtilis ◽

Structure Analysis ◽

Crystal Structure Analysis ◽

Structural Basis ◽

Substrate Selectivity

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Identification of a Critical Ankyrin-binding Loop on the Cytoplasmic Domain of Erythrocyte Membrane Band 3 by Crystal Structure Analysis and Site-directed Mutagenesis

Journal of Biological Chemistry ◽

10.1074/jbc.m211137200 ◽

2002 ◽

Vol 278 (9) ◽

pp. 6879-6884 ◽

Cited By ~ 58

Author(s):

Seon Hee Chang ◽

Philip S. Low

Keyword(s):

Crystal Structure ◽

Erythrocyte Membrane ◽

Structure Analysis ◽

Cytoplasmic Domain ◽

Band 3 ◽

Crystal Structure Analysis ◽

Site Directed Mutagenesis ◽

Directed Mutagenesis ◽

Binding Loop

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Structure of the processive human Pol δ holoenzyme

10.1101/872879 ◽

2019 ◽

Author(s):

Claudia Lancey ◽

Muhammad Tehseen ◽

Vlad-Stefan Raducanu ◽

Fahad Rashid ◽

Nekane Merino ◽

...

Keyword(s):

Dna Polymerase ◽

Proliferating Cell Nuclear Antigen ◽

Nuclear Antigen ◽

Structural Basis ◽

Catalytic Core ◽

Regulatory Subunits ◽

Functional Interactions ◽

Proliferating Cell ◽

Lagging Strand ◽

Catalytic Cleft

In eukaryotes, DNA polymerase δ (Pol δ) bound to the proliferating cell nuclear antigen (PCNA) replicates the lagging strand and cooperates with flap endonuclease 1 (FEN1) to process the Okazaki fragments for their ligation. We present the high-resolution cryo-EM structure of the human processive Pol δ-DNA-PCNA complex in the absence and presence of FEN1. Pol δ is anchored to one of the three PCNA monomers through the C-terminal domain of the catalytic subunit. The catalytic core sits on top of PCNA in an open configuration while the regulatory subunits project laterally. This arrangement allows PCNA to thread and stabilize the DNA exiting the catalytic cleft and recruit FEN1 to one unoccupied monomer in a toolbelt fashion. Alternative holoenzyme conformations reveal important functional interactions that maintain PCNA orientation during synthesis. This work sheds light on the structural basis of Pol δ’s activity in replicating the human genome.

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Classification of a VUS in MLH1 using a combination of family segregation studies and protein biochemistry.

Journal of Clinical Oncology ◽

10.1200/jco.2013.31.4_suppl.342 ◽

2013 ◽

Vol 31 (4_suppl) ◽

pp. 342-342

Author(s):

Kevin M. Zbuk ◽

Kathleen Bell ◽

Anna Zhou ◽

Alba Guarne ◽

Melyssa Aronson ◽

...

Keyword(s):

Crystal Structure ◽

Missense Variant ◽

Site Directed Mutagenesis ◽

Multiple Sources ◽

Dimerization Domain ◽

Protein Biochemistry ◽

Dna Mismatch ◽

Terminal Domain ◽

Amsterdam Criteria

342 Background: The identification of variants of unknown clinical significance (VUS) presents a challenge for diagnostic laboratories and clinicians working with families with Lynch syndrome (LS). Although a variety of strategies exist to attempt clarification, data from multiple sources is often required before a VUS is re-classified to pathogenic or benign. Methods: We report on the investigation of 2 families with a variant in the MMR gene MLH1, c.2038T>C (p.Cys680Arg). In addition to segregation analysis, and review of published literature, the c-terminal domain of MLH1was cloned, the p.Cys680Arg variant was generated using site-directed mutagenesis and protein expression was induced. In addition, this point mutation was modelled onto the crystal structure of the dimerization domain of MLH1. Results: Both families fit Amsterdam criteria for a diagnosis of LS. All available tumours exhibited MLH1 deficiency and microsatellite instability and all living affected individuals available were positive for MLH1 p.Cys680Arg variant. Protein studies confirmed that p.Cys680 resides in a tight hydrophobic cavity and the p.Cys680Arg mutation disrupts the folding of the C-terminal domain of MLH1. In two families this variant segregated with disease in a total of 12 individuals (>10 meiosis and >1000:1 odds in favor of linkage) and 3 had an MLH1 deficient tumour, supporting a pathogenic role for this variant. Inspection of the crystal structure of MLH1 suggested that the Cys680Arg mutation should severely alter the folding of the dimerization domain. As opposed to the dimerization domain of MLH1 that can be readily produced recombinantly and is stable in solution, the same domain of MLH1 encoding the Cys680Arg mutation was expressed as inclusion bodies, confirming that this mutation causes misfolding of the dimerization domain of MLH1. Conclusions: Our data confirms that the p.Cys680Arg mutation destabilizes the dimerization domain of MLH1, and presumably the DNA mismatch repair proficiency of individuals carrying this mutation. Based on this evidence and family history data, the MLH1 C680R missense variant is classified as pathogenic. This approach may prove useful in the classification of other variants.

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